1. Field of the Invention
The invention relates to recombinant mutants of the peptide lycotoxin-1, and the use of these peptides for insect and microbial control, transmembrane carrier tags, and expression level enhancer tags.
2. Description of the Prior Art
It is estimated that insect pests destroy 20-30% of the world's crop production [Oerke. In Crop Production and Crop Protection—Estimated Losses in Major Food and Cash Crops, Oerke et al. (eds). Elsevier: Amsterdam; 1994]. In many cases, natural enemies are not sufficient to control pests adequately [Hopper. United States department of agriculture—Agricultural Research Service research on biological control of arthropods. Pest Manag. Sci. 2003; 59(6-7): 643-653]. The Bacillus thuringiensis (Bt) crystal protein is one example of an insecticidal protein that has been developed commercially. It is highly effective against a targeted range of species depending on the clone [Hussein et al. Beetle-specific Bacillus thuringiensis Cry3Aa toxin reduces larval growth and curbs reproduction in Spodoptera littoralis (Boisd.). Pest Manag. Sci. 2005; 61: 1186-1192. and Herrero et al. Bacillus thuringiensis CryICa-resistant Spodoptera exigua lacks expression of one of four aminopeptidase N genes. BMC Genomics 2005; 6: 96] and has been employed in plants through transgenic production [Lambert et al. A Bacillus thuringiensis insecticidal crystal protein with a high activity against members of the family Noctuidae. Appl. Environ. Microbiol. 1996; 62(1): 80-86. Buntin et al. Plant-incorporated Bacillus thuringiensis resistance for control of fall armyworm and corn earworm (Lepidoptera: Noctuidae) in corn. J. Econ. Entomol. 2004; 97(5): 1603-1611. and Dowd et al. Strategies for insect management targeted toward mycotoxin management. In Aflatoxin and Food Safety, Abbas (ed.). Marcel Dekker: New York, 2005; 517-541] for complete control of some insect species, such as European corn borers [Peairs. 2002, 2003, 2006, 2007; (last update Sep. 24, 2007). Managing corn pests with Bt corn. Colorado State University Extension Bulletin No. 0.708, www.ext.colostate.edu/Pubs/crops/00708.html]. With commercialization of transgenic crops expressing Bt toxins, selection pressure has increased and there is concern that target insects may develop resistance to individual Bt proteins, requiring structured refuges of non-Bt host plants where Bt hybrids are grown [Peairs, ibid]. Even with such resistance management strategies, there is still concern that resistance will develop in insect pests receiving sublethal doses [Siegfried et al. Baseline susceptibility of western corn rootworm (Coleoptera: Crysomelidae) to Cry3Bb1 Bacillus thuringiensis toxin. J. Econ. Entomol. 2005; 98(4): 1320-1324], and cross-resistance to different Bt proteins has been reported [Ferr´ e and Van Rie. Biochemistry and genetics of insect resistance to Bacillus thuringiensis. Annu. Rev. Entomol. 2002; 47: 501-533]. Combinations of insecticidal protein genes may be needed for more durable control [Walker et al. A QTL that enhances and broadens Bt insect resistance in soybean. Theor. Appl. Genet. 2004; 109: 1051-1057]. Both synthetic materials [Nauen R, Smagghe G. Mode of action of etoxazole. Pest Manag. Sci. 2006; 62: 379-382] and naturally derived plant extracts [C´ espedes et al. Insect growth regulatory effects of some extracts and sterols from Myrtillocactus geometrizans (Cactaceae) against Spodoptera frugiperda and Tenebrio molitor. Phytochemistry 2005; 66: 2481-2493] continue to be explored as alternatives to Bt proteins.
The peptides in spider venoms appear to have potential for insect control owing to their specificity for the insect nervous system [Tedford et al. Scanning mutagenesis of omega-atracotoxin-Hv1a reveals a spatially restricted epitope that confers selective activity against insect calcium channels. J. Biol. Chem. 2004; 279(42): 44133-44140. and Wang et al. Discovery and structure of a potent and highly specific blocker of insect calcium channels. J. Biol. Chem. 2001; 276(43): 40306-40312]. Toxins from spider venoms define new insecticide targets owing to specific action to block insect voltage-gated Ca2+ channels [Nicholson and Graudins. Spiders of medical importance in the Asia-Pacific: atracotoxin, latrotoxin and related spider neurotoxins. Clin. Exp. Pharmacol. Physiol. 2002; 29(9): 785-794]. These toxins show promise for development of recombinant biopesticides for control of insecticide-resistant agricultural pests [Nicholson and Graudins, ibid]. Sequential alanine substitutions along the peptide chain of the insect-specific toxins of the funnel-web spiders in which each amino acid residue is separately replaced with alanine demonstrated critical features necessary for insecticidal activity of the toxins [Tedford et al. 2004, ibid. and Tedford et al. Functional significance of the β-hairpin in the insecticidal neurotoxin omega-atracotoxin-Hv1a. J. Biol. Chem. 2001; 276(28): 26568-26576]. A more efficient method for producing large numbers of toxin variants that are potentially more effective against insects would be to substitute each amino acid in the toxin peptide with all 20 possible amino acids rather than just one.
The selective spider venom peptide, lycotoxin-1 (also known as lycotoxin-I), from wolf spider (Lycosa carolinensis) venom, is a small 25 amino acid peptide that demonstrates both antimicrobial and insect neuroactive properties [Yan and Adams. Lycotoxins, antimicrobial peptides from venom of the wolf spider Lycosa carolinensis. J. Biol. Chem. 1998; 273(4): 2059−2066. and Kourie and Shorthouse. Properties of cytotoxic peptide-formed ion channels. Am. J. Physiol. Cell Physiol. 2000; 278: C1063-C1087], which from its amphipathic nature and physiological actions appears to function as a pore former to increase membrane permeability, dissipate voltage gradients, and effect lysis of insect cells. Antimicrobial peptides for a related species of Lycosa indicate that some variation in peptide sequence is possible without losing biological activity [Budnik et al. De novo sequencing of antimicrobial peptides isolated from the venom glands of the wolf spider Lycosa singoriensis. J. Mass Spectrom. 2004; 39(2): 193-201].